Search

We investigated the impairment of ATP-sensitive K+ (KATP) channels in aortic smooth muscle cells (ASMCs) from isoproterenol-induced hypertrophied rabbits. The amplitude of KATP channels induced by the KATP channel opener pinacidil (10 μM) was greater in ASMCs from control than from hypertrophied animals. In phenylephrine-preconstricted aortic rings, pinacidil induced relaxation in a dose-dependent manner. The dose-dependent curve was shifted to the right in the hypertrophied (EC50: 17.80 ± 3.28 μM) compared with the control model (EC50: 6.69 ± 2.40 μM). Although the level of Kir6.2 subtype expression did not differ between ASMCs from the control and hypertrophied models, those of the Kir6.1 and SUR2B subtypes were decreased in the hypertrophied model. Application of the calcitonin-gene related peptide (100 nM) and adenylyl cyclase activator forskolin (10 μM), which activates protein kinase A (PKA) and consequently KATP channels, induced a KATP current in both control and hypertrophied animals; however, the KATP current amplitude did not differ between the two groups. Furthermore, PKA expression was not altered between the control and hypertrophied animals. These results suggests that the decreased KATP current amplitude and KATP channel-induced vasorelaxation in the hypertrophied animals were attributable to the reduction in KATP channel expression but not to changes in the intracellular signaling mechanism that activates the KATP current.

In this study, dynamic changes in nitric oxide (NO) and mitochondrial superoxide (O2•−) were examined during anoxic preconditioning (AP) in rat heart model. AP and anoxia-reoxygenation (A/R) were performed on isolated hearts and single cardiomyocytes. The cellular insult in the form of infarct size and DNA damage were localized and correlated with NO synthases (endothelial and inducible) expression levels. The results showed that endocardium was the most affected region in AP groups, whereas the larger area of infarct was confined to mid- and epicardium in the A/R group. Interestingly, a high-level expression of immunofluorescent NO synthases was restricted to viable areas in the AP. In contrast to the gradual increase in O2•− level that occurred in the AP group, a sudden massive increase in its level was demonstrated at the onset of reoxygenation in the A/R group. The observed increase in NO production during reoxygenation in the AP group was attenuated by inducible NO synthase inhibitor. The study revealed, on a real-time basis, the role played by preconditioning for modulating NO and O2•− levels on behalf of cell survival. The results afford a better understanding of cardiac-adapting mechanism during AP and the role of inducible NO synthase in this important phenomenon.

We investigated the role of the mitochondrial ATP-sensitive K+(KATP) channel, the mitochondrial big-conductance Ca2+-activated K+(BKCa) channel, and the mitochondrial permeability transition pore (MPTP) in the ouabain-induced increase of mitochondrial Ca2+in native rat ventricular myocytes by loading cells with rhod 2-AM. To overload mitochondrial Ca2+, we pretreated cells with ouabain before applying mitochondrial KATPor BKCachannel and/or MPTP opener. Ouabain (1 mM) increased the rhod 2-sensitive fluorescence intensity (160 ± 5.0% of control), which was dramatically decreased to the control level on application of diazoxide and NS-1619 in a dose-dependent manner (half-inhibition concentrations of 78.3 and 7.78 μM for diazoxide and NS-1619, respectively). This effect was reversed by selective inhibition of the mitochondrial KATPchannel by 5-hydroxydecanoate, the mitochondrial BKCachannel by paxilline, and the MPTP by cyclosporin A. Although diazoxide did not efficiently reduce mitochondrial Ca2+during prolonged exposure to ouabain, NS-1619 reduced mitochondrial Ca2+. These results suggest that although mitochondrial BKCaand KATPchannels contribute to reduction of ouabain-induced mitochondrial Ca2+overload, activation of the mitochondrial BKCachannel more efficiently reduces ouabain-induced mitochondrial Ca2+overload in our experimental model.

We examined the effects of acute hypoxia on Ba2+-sensitive inward rectifier K+(KIR) current in rabbit coronary arterial smooth muscle cells. The amplitudes of KIRcurrent was definitely higher in the cells from small-diameter (200 μm, LCASMC, −1.5 ± 0.1 pA pF−1). Western blot analysis confirmed that Kir2.1 protein was expressed in SCASMC but not LCASMC. Hypoxia activated much more KIRcurrents in symmetrical 140 K+. This effect was blocked by the adenylyl cyclase inhibitor SQ-22536 (10 μM) and mimicked by forskolin (10 μM) and dibutyryl-cAMP (500 μM). The production of cAMP in SCASMC increased 5.7-fold after 6 min of hypoxia. Hypoxia-induced increase in KIRcurrents was abolished by the PKA inhibitors, Rp-8-(4-chlorophenylthio)-cAMPs (10 μM) and KT-5720 (1 μM). The inhibition of G protein with GDPβS (1 mM) partially reduced (∼50%) the hypoxia-induced increase in KIRcurrents. In Langendorff-perfused rabbit hearts, hypoxia increased coronary blood flow, an effect that was inhibited by Ba2+. In summary, hypoxia augments the KIRcurrents in SCASMC via cAMP- and PKA-dependent signaling cascades, which might, at least partly, explain the hypoxia-induced coronary vasodilation.

In freshly isolated rabbit pulmonary artery smooth muscle cells, endothelin (ET)-1 induced a transient increase in intracellular Ca2+concentration ([Ca2+]i) followed by a return to the initial [Ca2+]i. This response was not abolished by the voltage-dependent Ca2+channel blocker nicardipine or removal of Ca2+from the bath solution but was inhibited by ryanodine and thapsigargin. This finding suggested that the increase in [Ca2+]iinduced by ET-1 was attributable to release of Ca2+from ryanodine- and inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+stores. The transient increase in [Ca2+]iinduced by ET-1 was also inhibited by pretreatment with antagonists of ET type A and B (ETAand ETB) receptors (BQ-123 and BQ-788, respectively). Furthermore, the ETBreceptor agonist IRL-1620 induced an increase in [Ca2+]ithat was followed by a sustained increase in [Ca2+]i; the sustained increase in [Ca2+]iwas blocked by nicardipine. Using the nystatin-perforated patch-clamp technique, we found that IRL-1620 caused an increase in Ca2+current that was inhibited by addition of ET-1. ET-1 did not inhibit Ca2+current when cells were pretreated with BQ-123. These results suggested that when both receptor types are activated, the opposing responses lead to abolition of the sustained [Ca2+]iincreases induced by ETBreceptor activation. Western blot analysis confirmed expression of ETAand ETBreceptors. Finally, U-73122 inhibited the ET-1-induced [Ca2+]iincrease, indicating that phospholipase C was involved in modulation of the ET-1-induced [Ca2+]iincrease in rabbit pulmonary artery smooth muscle cells.

Human adipose tissue-derived mesenchymal stem cells (hASCs) have the power to differentiate into various cell types including chondrocytes, osteocytes, adipocytes, neurons, cardiomyocytes, and smooth muscle cells. We characterized the functional expression of ion channels after transforming growth factor-β1 (TGF-β1)-induced differentiation of hASCs, providing insights into the differentiation of vascular smooth muscle cells. The treatment of hASCs with TGF-β1 dramatically increased the contraction of a collagen-gel lattice and the expression levels of specific genes for smooth muscle including α-smooth muscle actin, calponin, smooth mucle-myosin heavy chain, smoothelin-B, myocardin, and h-caldesmon. We observed Ca2+, big-conductance Ca2+-activated K+ (BKCa), and voltage-dependent K+ (Kv) currents in TGF-β1-induced, differentiated hASCs and not in undifferentiated hASCs. The currents share the characteristics of vascular smooth muscle cells (SMCs). RT-PCR and Western blotting revealed that the L-type (Cav1.2) and T-type (Cav3.1, 3.2, and 3.3), known to be expressed in vascular SMCs, dramatically increased along with the Cavβ1 and Cavβ3 subtypes in TGF-β1-induced, differentiated hASCs. Although the expression-level changes of the β-subtype BKCa channels varied, the major α-subtype BKCa channel (KCa1.1) clearly increased in the TGF-β1-induced, differentiated hASCs. Most of the Kv subtypes, also known to be expressed in vascular SMCs, dramatically increased in the TGF-β1-induced, differentiated hASCs. Our results suggest that TGF-β1 induces the increased expression of vascular SMC-like ion channels and the differentiation of hASCs into contractile vascular SMCs.